Sphingosine-1-phosphate (SPP) is a bioactive sphingolipid metabolite which regulates diverse biological processes (reviewed in (Goetzl, et al., (1998) FASEB J.12, 1589-1598 and Spiegel, S. (1999) J. Leukoc. Biol. 65, 341-344.) Many of its actions are reported to be mediated by a family of specific cell surface G-protein coupled receptors (GPCR), known as EDG (endothelial differentiation genes) receptors. Binding of SPP to EDG-1 expressed on endothelial cells reportedly enhances survival (Hisano, et al., (1999) Blood 93, 4293-4299), chemotaxis and in vitro angiogenesis (Wang, et al., (1999) J. Biol. Chem. 274, 35343-35350) and adherens junction assembly leading to morphogenetic differentiation (Lee, et al., (1999) Cell 99, 301-312), whereas binding of SPP to EDG-5 and EDG-3 is reported to induce neurite retraction and soma rounding (Postma, et al., (1996) EMBO J. 15, 2388-2392 and Van Brocklyn, et al., (1999) J. Biol. Chem. 274, 46264632). Additional research indicates that SPP induces activation of Gi-gated inward rectifying K+-channels in atrial myocytes (van Koppen, et al., (1996) J. Biol. Chem. 271, 2082-2087) and inhibits motility of melanoma cells (Yamamura, et al., (1997) Biochemistry 36, 10751-10759) through as yet uncharacterized GPCRs.
SPP is also described as performing important roles inside cells. In response to diverse external stimuli, sphingosine kinase, the enzyme that catalyzes the phosphorylation of sphingosine to SPP, is activated (Olivera, et al., (1993) Nature 365, 557-560; Choi, et al., (1996) Nature 380, 634-636; Melendez, et al., (1998) J. Biol. Chem. 273, 9393-9402; Xia, et al., (1998) Proc. Natl. Acad. Sci. USA 95, 14196-14201; Kleuser, et al., (1998) Cancer Res. 58, 1817-1824 and Meyer zu Heringdorf, et al., (1998) EMBO J. 17, 2830-2837). Intracellular SPP in turn mobilizes calcium from internal stores independently of InsP3 (Meyer zu Heringdorf, et al., (1998) EMBO J. 17, 2830-2837 and Mattie, et al., (1994) J. Biol. Chem. 269, 3181-3188), as well as eliciting diverse signaling pathways leading to proliferation (Rani, et al., (1997) J. Biol. Chem. 272, 10777-10783 and Van Brocklyn, et al., (1998) J. Cell Biol. 142, 229-240.) and suppression of apoptosis (Cuvillier, et al., (1996) Nature 381, 800-803; Perez, et al., (1997) Nature Med. 3, 1228-1232; Edsall, et al., (1997) J. Neurosci. 17, 6952-6960; Cuvillier, et al., (1998) J. Biol. Chem. 273, 2910-2916).
Because of its dual function as a ligand and second messenger and its pivotal role in cell growth and survival, the synthesis and degradation of SPP is expected to be tightly regulated in a spatial-temporal manner. Until recently, however, little was known of the enzymes involved in SPP metabolism. A previous report described the purification of sphingosine kinase to apparent homogeneity from rat kidney (Olivera, et al., (1998) J. Biol. Chem. 273, 12576-12583). Subsequently the first mammalian sphingosine kinase was cloned from rat and characterized (Kohama, et al., (1998) J. Biol. Chem. 273, 23722-23728). The kinase is described as belonging to a novel, highly conserved gene family (Kohama, et al., (1998) J. Biol. Chem. 273, 23722-23728 and Nagiec, et al., (1998) J. Biol. Chem. 273, 19437-19442). Enforced expression of the sphingosine kinase markedly enhanced the proliferation and survival of cells, substantiating the importance of intracellularly generated SPP in cell fate decisions (Olivera, et al., (1999) J. Cell Biol. 147, 545-548).
SPP can be metabolized by two distinct pathways. In one pathway, SPP is catabolized via a microsomal pyridoxal phosphate-dependent lyase to palmitaldehyde and phosphoethanolamine, which can then be utilized for the biosynthesis of glycerolipids. In a second pathway, SPP is dephosphorylated by specific phosphatases to sphingosine (Spiegel, et al., (1996) FASEB J. 10, 1388-1397).
Genetic manipulation studies in yeast have demonstrated an important role for long-chain phosphorylated sphingoid bases in growth and survival of yeast after nutrient deprivation and heat stress (Mandala, et al., (1998) Proc. Nat. Acad. Sci. USA 95, 150-155; Gottlieb, et al., (1999) Mol. Cell Biol. Res. Commun. 1, 66-71; Mao, et al., (1999) Biochem. J. 342, 667-675 and Skrzypek, et al., (1999) J. Bacteriol. 181, 1134-1140) in a manner which is reminiscent of their effects on mammalian cells. Recently, the yeast genes encoding the lyase and phosphatase enzymes of these two catabolic pathways were identified in S. cerevisiae (Saba, et al. (1997) J. Biol. Chem. 272, 26087-26090; Mandala, et al., (1998) Proc. Nat. Acad. Sci. USA 95, 150-155 and Mao, et al., (1997) J. Biol. Chem. 272, 28690-28694).
The yeast SPP phosphatases encoded by LBP1 and LBP2 are members of Type 2 lipid phosphate phosphohydrolases, a family of magnesium independent, membrane-bound enzymes that share sequence conservation within three domains that are predicted to be involved in the coordination and hydrolysis of the phosphate moiety (Stukey, et al., (1997) Protein Sci. 6, 469-472). A search of the yeast genome for enzymes containing the three conserved domains revealed the presence of 4 genes encoding putative Type 2 lipid phosphatases. Two of these, DPP1 and LPP1, were shown to encode phosphatases with activity against phosphatidic acid (PA), lysophosphatidic acid (LPA), and diacylglycerol pyrophosphate (DGPP) (Toke, et al., (1998) J. Biol. Chem. 273, 14331-14338 and Toke, et al., (1998) J. Biol. Chem. 273, 3278-3284). In contrast, LBP1 (also known as YSR2 or LCB3) and LBP2 (YSR3), encode phosphatases with remarkable specificity for phosphorylated sphingoid bases and without activity towards glycerolipid substrates (Mandala, et al., (1998) Proc. Nat. Acad. Sci. USA 95, 150-155; Mao, et al., (1997) J. Biol. Chem. 272, 28690-28694 and Skrzypek, et al., (1999) J. Bacteriol. 181, 1134-1140).
The presence of a high affinity SPP phosphatase activity with enzymatic properties similar to yeast SPP phosphatases has been described in crude rat liver and cerebellum extracts (De Ceuster, et al., (1995) Biochem. J. 311, 139-146). Although three isoforms of Type 2 lipid phosphate phosphohydrolases, known as LPP1/PAP2a, LPP3/PAP2b, and LPP2/PAP2c, have been cloned from mammalian cells (reviewed in (Brindley, et al., (1998) J. Biol. Chem. 273, 24281-24284)), these gene products appear to have broad substrate specificity with similar efficiencies against PA, LPA, SPP, ceramide-1-P, and DGPP, when assayed in vitro in lipid/detergent micelles.